Fig 1.
Diagram of the experimental procedure used and the analysis performed on L. bulgaricus CFL1 samples.
DSC, FITR, viability and acidification activity measurements were performed on at least three independent biological replicates. Steps involving different conditions are in blue. Similar experimental procedure was used for S. cervisiae 338.
Fig 2.
DSC traces of S. cerevisiae 338 cells during cooling.
(a) DSC heat flow traces at 50°C.min-1 (red curves), 10°C.min-1 (green curves) and 2°C.min-1 (blue curves) and (b) DSC first derivative of the heat flow during warming at 10°C.min-1. In all figures the effects of a repeat freeze thaw were also determined. Capital letters A, B and C in Fig 2A correspond to exothermic events indicated in Table 1.
Table 1.
Summary of the thermal events observed by DSC with S. cerevisiae 338 following freezing at different cooling rates.
Fig 3.
CryoSEM of S. cerevisiae 338 cells following cooling.
(a) At 50°C.min-1 and (b) at 2°C.min-1. Note the large voids in cells cooled at 50°C.min1, caused by the formation of intracellular ice.
Table 2.
Intracellular vitrification temperature for L. bulgaricus CFL1 following growth under different conditions, following the addition of cryoprotective additive and subsequent wash (Tg’) determined by DSC.
Fig 4.
DSC first derivative of heat flow of L. bulgaricus CFL1 with cryoprotective additives and aqueous solutions of cryoprotectants.
(a) sucrose, (b) glycerol and (c) DMSO are all additives at 0.58 M. The vitrification temperatures of incubated (light blue curve) and subsequently washed cells (grey curve) are indicated. Samples were cooled below −90°C and then warmed at 10°C.min-1. Control samples (washed with peptone water) (black curves) and aqueous solutions of cryoprotectants (dark blue curves) are also presented for comparison.
Fig 5.
The effects of cryoprotective agents on the loss of specific acidification activity and viability upon freeze-thawing of L. bulgaricus CFL1.
Cells were grown in either whey or MRS medium, harvested in stationary culture phase and frozen to -80°C at 2°C min-1 with sucrose, glycerol or DMSO (all additives at 0.58 M) and no additive (control: 1 g.L-1 peptone water). Superscripts letters represent statistical contrasts (significant differences) between samples at the 95% confidence level. Control whey grown cells displayed an extreme loss in cell function (64.8 ± 3.5 (min.log(CFU.mL-1)-1)) and viability upon thawing (3.3 ± 0.3 log(CFU.mL-1)), and for clarity are not included in this figure.
Fig 6.
The loss of specific acidification activity on thawing of L. bulgaricus plotted against intracellular vitrification temperature (Tg’).
Cells were grown in either whey (dark grey) or MRS medium (light grey), harvested in stationary culture phase and frozen to -80°C at 2°C min-1 with sucrose (triangles), glycerol (diamonds) or DMSO (squares), (all additives at 0.58 M) and MRS control (circle). Dotted arrow indicates the direction of control whey grown cells loss in cell function (64.8 ± 3.5 (min.log(CFU mL-1)-1)).
Fig 7.
Schematic of the transitions which occur during cryopreservation of L. bulgaricus CFL1.
Temperature transitions correspond to cells grown in whey medium and protected with glycerol (0.58 M).